New alpha-amino phenylalanine tetrazole ligand for immobilized metal affinity chromatography of proteins

A new chelating compound has been developed for use in the immobilized metal affinity chromatographic (IMAC) separation of proteins. The bidentate ligand, alpha-amino phenylalanine tetrazole, 4, was synthesized via a five-step synthesis from N-fluorenylmethoxycarbonyl phenylalanine and then immobilized onto silica through the epoxide coupling procedure. The binding behavior of the resulting IMAC sorbent, following chelation with Zn2+ to a density of 183 micromol Zn2+ ions/g silica, was characterized by the retention of proteins in the pH range of 5.0-8.0, and by the adsorption behavior of lysozyme with frontal chromatography at pH 6.0 and 8.0. The prepared column showed the separation ability to four test proteins and the retention time of these proteins increased with an increase in pH. From the derived isotherms, the adsorption capacity, qm, for the binding of lysozyme to immobilized Zn2+-alpha-amino phenylalanine tetrazole-silica was found to be 1.21 micromol/g at pH 6.0 and 1.20 micromol/g sorbent at pH 8.0, respectively, whilst the dissociation constants KD at these pH values were 5.22x10(-6) and 3.49x10(-6) M, respectively, indicating that the lysozyme was retained more stable under alkaline conditions, although the binding capacity in terms of micromole protein per gram sorbent remained essentially unchanged.     

Affinity Chromatography of Ribitol Dehydrogenase,a Possible New Zinc Enzyme from Mycobacterium Butyricum

Abstract

An effective purification procedure of ribitol dehydrogenase (RDH), a possible new zinc enzyme from Mycobacterium butyricum is described. The procedure took advantage of different chromatographic methods in which the most significant were two affinity chromatography steps. One of them was the immobilized metal ion affinity chromatography (IMAC), with the use of iminodiacetate-Sepharose 6B (IDA-Sepharose 6B) chelating Zn²⁺ ions (IDA-Zn) as an affinity sorbent. The enzyme was eluted with a decreasing pH gradient from 7 to 4. The other step was a biospecific affinity chromatography, where the enzyme retained on 5′ AMP-Sepharose 6B was eluted with 10 mM adenosine 5′-monophosphate (AMP). RDH was purified 174-fold with 10.2% of recovery, and the final preparation was homogenous in polyacrylamide gel electrophoresis.     

Purification of the specific immunoglobulin G1 by immobilized metal ion affinity chromatography using nickel complexes of chelating porous and nonporous polymeric sorbents based on poly(methacrylic esters). Effect of polymer structure

Ni2+ complexes of the chelating nonporous and porous bead sorbents based on methacrylic esters crosslinked with ethylene dimethacrylate were used in isolation of the horseradish peroxidase-specific immunoglobulin IgG1 from the crude mouse ascitic fluid by immobilized metal ion affinity chromatography (IMAC). Iminodiacetic and aspartic acids were attached to porous poly(glycidyl methacrylate) beads differing in size, morphology and chemical composition. Ethylenediaminetriacetic acid and quinolin-8-ol chelating groups were attached mainly to the surface hydroxyl groups in nonporous poly(diethylene glycol methacrylate) beads through spacers. The latter sorbents exhibited better kinetic characteristics than the former but a very low IgG1 sorption capacity. In a single-step IMAC procedure, the best efficiency in the specific IgG1 purification was obtained with porous sorbents (recovery 92%, purity 73%). Differences in IMAC separations are discussed from the point of view of morphology of polymer beads as well as of the type and concentration of chelating ligands.     

Immobilized Metal Affinity Chromatography of Phosphorylated Proteins Using High Performance Sorbents

The interactions of two model phosphoproteins (porcine pepsin and ovalbumin) with two different immobilized metal affinity chromatography (IMAC) sorbents containing immobilized Fe³⁺, Ga³⁺, and UO₂ ²⁺ ions have been investigated under various conditions. Both proteins were adsorbed on immobilized uranyl ions under acidic conditions similar to those on immobilized Fe³⁺ and Ga³⁺ ions. The retained proteins could be released either by the presence of phosphate ions in the elution buffer (immobilized Ga³⁺ and Fe³⁺ ions) or by an increased pH (all tested immobilized ions). The IMAC sorbents employed could be used under the conditions of high-performance chromatography and are suitable for the separation and analysis of intact phosphoproteins.     

The use of liquid phase deposition prepared phosphonate grafted silica nanoparticle‐deposited capillaries in the enrichment of phosphopeptides

In our current work, we describe how open tubular‐immobilized metal‐ion affinity chromatography (OT‐IMAC) capillary columns connected to a solid phase microextraction (in‐tube SPME) device can be used for the enrichment of phosphopeptides. A phosphonate modified silica nanoparticle (NP)‐deposited capillary was prepared by liquid phase deposition (LPD), and used for the immobilization of Fe³⁺, Zr⁴⁺ or Ti⁴⁺. The enrichment capacities of three different OT‐IMAC capillary columns were compared by using tryptically digested α‐casein as sample. The improved extraction efficiency in our technique was demonstrated by comparing to a directly modified capillary, and a comparison of phosphopeptide extraction from simple and complex samples was tested for both modes. Our results show that the NP‐IMAC‐Zr⁴⁺ capillary column can be used to selectively isolate phosphopeptides from real samples, and can enrich for β‐casein phosphopeptides from concentrations as low as 1.7×10^?9 M.     

固定金属离子亲和色谱--蛋白质分离的方法、原理、特性和应用

介绍了固定金属离子亲和色谱法(IMAC)的方法原理、金属螯合柱的制备、固定金属离子与蛋白质的相互作用以及影响这些作用的因素、不同色谱条件下各种作用力对蛋白质保留值的贡献、蛋白质的洗脱原理和IMAC在蛋白质分离纯化中的应用,论述了IMAC的特点、不足、克服的方法和今后应解决的问题。     

多齿配体-硅胶色谱介质的制备与评价

以新型环保多齿螯合剂——亚氨基二琥珀酸(IDS)为配体,在优化条件下,合成了IDS-Silica固定相。用电位滴定法测定了固定相上IDS的键合量。考察了IDS-Silica柱的色谱特性以及金属螯合特性。使用制备柱成功地分离了标准蛋白质混合物,该制备柱展现出了典型的阳离子交换特性。用电感耦合等离子体原子发射光谱法考察了金属离子在IDS-Silica固定相上的键合特性。结果表明,金属离子在IDS-Silica固定相上键合量的变化规律与它们同该固定相螯合的强弱顺序一致。通过比较金属Cu~(2+)在4种不同氨羧类配体硅胶柱上的键合量,发现IDS对金属离子具有强的螯合能力。IDS对金属离子的强螯合特性为其今后作为固定金属亲和色谱填料奠定了基础,为缓解亲和柱在使用过程中固定金属离子的流失提供了一种有效的解决方法。     

Preparation of Immobilized Metal Affinity Chromatographic Packings by Immobilization of Carboxymethylated Asparate (CM-Asp) Based on Monodisperse Hydrophilic Non-porous Beads and Their Application

The hydrophilic immobilized metal affinity chromatographic packing was prepared by immobilization of carboxymethylated asparate (CM‐Asp) as chelating ligand and Ni²⁺ as center ion on the base of monodispersed, 3.0 µm non‐porous monodisperse poly(glycidylmethacrylate‐co‐ethylenedimethacrylate) (P_GMA/EDMA) particles. The retention behavior of proteins and the effect of pH on the retention in the range from 4.0 to 9.0 were investigated on both the naked and metal ion chelated columns. Four proteins were quickly separated in 2.0 min with linear gradient elution at a flow rate of 3.0 mL·min^?1 by using the synthesized Ni²⁺‐CM‐Asp‐P_GMA/EDMA packings. The separation time was shorter than other immobilized metal affinity chromatography reported in the literature. The Ni²⁺‐CM‐Asp‐P_GMA/EDMA column was further investigated for the rapid separation and purification of copper‐zinc superoxide dismutase (Cu,Zn‐SOD) from the blood of pig in 3.0 min with only one step. The results obtained were satisfactory.     

Purification of Plasma-Derived Coagulation Factor VIII by Immobilized-Zn<Superscript>2+</Superscript> and -Co<Superscript>2+</Superscript> Affinity Chromatography

Coagulation factor VIII (FVIII) is a glycoprotein that plays a crucial role in the clotting cascade. Replacement therapies with recombinant and plasma-derived concentrates of FVIII are used for treatment of hemophilia A. We have previously purified the human plasma FVIII by immobilized metal affinity chromatography (IMAC) using Cu²⁺ as the metal ligand. In this work we report the purification of FVIII using Zn²⁺ and Co²⁺, two metal ions that bind proteins more weakly. Human plasma was directly applied to the anion-exchange ANX Sepharose FF column and the eluate was used as starting material for the studies in IMAC columns. Using imidazole as desorbing agent, FVIII was recovered with 65% activity in the IMAC-Zn²⁺ column and with 74% activity in the IMAC-Co²⁺ column. Purification factors were 4 and 9, respectively. Using a pH gradient, FVIII was eluted at pH 5.0 with 17% activity in the IMAC-Zn²⁺ and 77% activity in the IMAC-Co²⁺. Vitamin K-dependent proteins, a family of proteins that includes Prothrombin and coagulation factor IX, coeluted with FVIII in the ANX Sepharose FF column and were recovered with the unbound proteins on both IMAC columns. Therefore, Co²⁺ and Zn²⁺ columns were as effective as the Cu²⁺ column in separating FVIII from vitamin K-dependent proteins. Finally, we have shown that FVIII remained complexed with the von Willebrand factor.     

Properties and Application of a Chelating Sorbent Prepared by Modification of LiChroprep-NH<Subscript>2</Subscript> with Calcon

A new chelating sorbent for metal ions was prepared by modification of chemically modified silica – LiChroprep-NH₂ with Calcon. The molecular mechanism of binding this reagent to the surface of the applied carrier is presented. The properties of this sorbent were compared to analogous sorbents with a plain silica carrier and chemically modified silicas – LiChroprep-RP containing Calcon. The advantages of the new sorbent compared to the silica and LiChroprep-RP chelating sorbents are demonstrated. The sorbent obtained was applied as stationary phase in solid-phase extraction (SPE) for separations of some chosen mixtures of metal ions and for additional purification of aqueous solutions of salts of alkali metals from trace amounts of heavy metals. The multiple use of the sorbent based on LiChroprep-NH₂ in sorption-desorption processes of metal ions without deterioration of its sorption capacity is demonstrated.     

Use of phage display methods to identify heptapeptide sequences for use as affinity purification 'tags' with novel chelating ligands in immobilized metal ion affinity chromatography

This study describes the screening of a peptide phage display library for amino acid sequences that bind with different affinities to a novel class of chelating ligands complexed with Ni2+ ions. These chelating ligands are based on the 1,4,7-triazacyclononane (TACN) structure and have been chosen to allow enhanced efficiency in protein capture and decreased propensity for metal ion leakage in the immobilized metal ion affinity chromatographic (IMAC) purification of recombinant proteins. Utilising high stringency screening conditions, various peptide sequences containing multiple histidine, tryptophan, and/or tyrosine residues were identified amongst the different phage peptide sequences isolated. The structures, and particularly the conserved locations of these key amino acid residues within the selected heptapeptides, form a basis to design specific peptide tags for use with these novel TACN ligands as a new mode of IMAC purification of recombinant proteins.     

Efficient refolding of a hydrophobic protein with multiple S-S bonds by on-resin immobilized metal affinity chromatography

The efficient refolding of recombinant proteins produced in the form of inclusion bodies (IBs) in Escherichia coli still is a complicated experimental problem especially for large hydrophobic highly disulfide-bonded proteins. The aim of this work was to develop highly efficient and simple refolding procedure for such a protein. The recombinant C-terminal fragment of human alpha-fetoprotein (rAFP-Cterm), which has molecular weight of 26 kDa and possesses 6 S-S bonds, was expressed in the form of IBs in E. coli. The C-terminal 7× His tag was introduced to facilitate protein purification and refolding. The refolding procedure of the immobilized protein by immobilized metal chelating chromatography (IMAC) was developed. Such hydrophobic highly disulfide-bonded proteins tend to irreversibly bind to traditionally used agarose-based matrices upon attempted refolding of the immobilized protein. Indeed, the yield of rAFP-Cterm upon its refolding by IMAC on agarose-based matrix was negligible with bulk of the protein irreversibly stacked to the resin. The key has occurred to be using IMAC based on silica matrix. This increased on-resin refolding yield of the target protein from almost 0 to 60% with purity 98%. Compared to dilution refolding of the same protein, the productivity of the developed procedure was two orders higher. There was no need for further purification or concentration of the renatured protein. The usage of silica-based matrix for the refolding of immobilized proteins by IMAC can improve and facilitate the experimental work for difficult-to-refold proteins.     

The impact of immobilized metal affinity chromatography (IMAC) resins on DNA aptamer selection

DNA aptamers are single-stranded oligonucleotides which can form various secondary and tertiary structures. They can recognize a broad range of targets ranging from small molecules, such as ions, vitamins, antibiotics, to high molecular weight structures, including enzymes and antibodies. DNA aptamers are extensively studied as a potential source of new pharmaceutical drugs due to their inexpensive synthesis, low immunogenicity, and high specificity. The commonly used aptamer selection procedure is systematic evolution of ligands by exponential enrichment (SELEX) where the target molecule is immobilized on an appropriate chromatography resin. For peptide/protein targets, immobilized metal affinity chromatography (IMAC) resins are frequently used. There is a broad range of commercially available resins which can be used for IMAC. They are characterized by different metal ions, linker types, and bead materials. In this study, we tested the impact of different IMAC resins on the DNA aptamer selection process during eight SELEX cycles. A histidine-tagged 29 amino acid peptide corresponding to the interdomain connecting loop of human proliferating cell nuclear antigen was used as a selection target. Different resin materials containing the same metal ion (Co²⁺) were tested. Simultaneously, agarose resins containing identical linkers, but different metal ions (Co²⁺, Cu²⁺, Ni²⁺, and Zn²⁺) were analyzed. The results of this study clearly demonstrated the impact of the metal ion and resin material on the DNA aptamer selection progress. The presented data indicate that for successful IMAC resin-based SELEX, the determination of the optimal resin might be crucial.     

High-Speed Purification of Recombinant Interleukin-11 by IMAC with Rigid Biporous Beads

Rigid biporous beads were prepared and modified by iminodiacetic acid (IDA) for application in immobilized metal affinity chromatography of proteins. The retention behavior of four model proteins on the metal chelate columns loaded with copper (II) and nickel (II) ions were studied. The separation of the four proteins by the Ni-IDA column at 40 cm.min⁻¹ was realized within 2 min. His6-interluekin-11 (His6-IL-11) was also purified by the Ni-IDA column at 40 cm.min⁻¹. The collected His6-IL-11 fraction showed a purity of about 80%. The results indicate that the IMAC with the biporous medium is promising for high-speed protein purification.     

Development of new multifunctional terpolymer sorbent for proteomics applications

Determination of the availability of phases for specific separations is an important task achieved by a separation chemist. This becomes vital when the complex samples like biofluids are dealt with in proteome science. The work presented here involves the synthesis and application of terpolymeric sorbent with different functionalizations adopted for the selective enrichment of biomolecules of interest from biological fluids. Synthesis of terpolymer was carried out by the radical polymerization of monomers: methyl acrylate, acrylic acid and vinyl acetate with diethylene glycol dimethacrylate as cross‐linking agent, benzoyl peroxide as initiator and chloroform as a porogenic solvent. Characterization was done through Fourier transform infrared spectroscopy, scanning electron microscopy and nitrogen adsorption porosimetry. The polymer was further modified to immobilized metal ion affinity chromatographic material, with immobilized Fe³⁺/La³⁺ ions that allowed phosphopeptide enrichment from tryptic digests of standard proteins as well as milk, egg yolk and human serum. Sensitivity of enrichment down to 50 fmol was achieved in the presence of complex protein background as bovine serum albumin. Hydrophobicity was introduced through octadecyl amine, which provides comparable results to ZipTip C₁₈/C₄ for desalting of complex mixtures of all caseins. Analysis of the enriched content was performed by Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI‐MS). Copyright © 2014 John Wiley & Sons, Ltd.     

Chromatographic behaviour of monoclonal antibodies against wild-type amidase from Pseudomonas aeruginosa on immobilized metal chelates

The aim of this work was to devise a one‐step purification procedure for monoclonal antibodies (MAbs) of IgG class by immobilized metal affinity chromatography (IMAC). Therefore, several stationary phases were prepared containing immobilized metal chelates in order to study the chromatographic behaviour of MAbs against wild‐type amidase from Pseudomonas aeruginosa. Such MAbs adsorbed to Cu(II), Ni(II), Zn(II) and Co(II)–IDA agarose columns. The increase in ligand concentration and the use of longer spacer arms and higher pH values resulted in higher adsorption of MAbs into immobilized metal chelates. The dynamic binding capacity and the maximum binding capacity were 1.33 ± 0.015 and 3.214 ± 0.021 mg IgG/mL of sedimented commercial matrix, respectively. A K_D of 4.53 × 10⁻⁷ m was obtained from batch isotherm measurements. The combination of tailor‐made stationary phases of IMAC and the correct selection of adsorption conditions permitted a one‐step purification procedure to be devised for MAbs of IgG class. Culture supernatants containing MAbs were purified by IMAC on commercial‐Zn(II) and EPI‐30–IDA–Zn(II) Sepharose 6B columns and by affinity chromatography on Protein A‐Sepharose CL‐4B. This MAb preparation revealed on SDS–PAGE two protein bands with M_r of 50 and 22 kDa corresponding to the heavy and light chains, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Synthesis of silica nanospheres with Ni2+-iminodiacetic acid for protein separation

Silica (SiO₂) nanospheres (NSs) with immobilized metal ligands have been prepared for the affinity separation of proteins. First, SiO₂ NSs were prepared by controlled hydrolysis of tetraethoxysilane in a basic aqueous-ethanol solution. Then through reaction of iminodiacetic acid (IDA) with 3-glycidoxypropyltrimethoxysilane and immobilization of them onto the surfaces of above SiO₂ NSs, novel affinity adsorbents with IDA chelating groups were obtained. After chelating Ni²⁺ ions, the SiO₂–IDA–Ni²⁺ NSs were applied to separate his-tagged proteins directly from the mixture of lysed cells. The SiO₂–IDA–Ni²⁺ NSs present negligible nonspecific protein adsorption and high protein binding ability (28.3 mg/g).     

Separation of chitosan oligomers by immobilized metal affinity chromatography

A novel approach for chitosan oligosaccharide (COS) separation by immobilized metal affinity chromatography (IMAC) based on the differences in the interactions of chelated copper (II) ions with various COS (dimers, trimers, tetramers) is described. Polyhydroxylic chromatographic supports (agarose CL-6B and silica) were functionalized with various chelating functions such as iminodiacetate (IDA), carboxymethyl-aspartate (CM-Asp) and tris(carboxymethyl)ethylenediamine (TED). The COS retention capacities of the columns were between 2 and 6 mg/cm(3), depending on the chelating group. The COS were separated and/or enriched up to 95% for dimer and trimer and 90% for the tetramer, with yields of 60-95%.     

Enhancement of Mercury Sorbent Using Metal Aluminate Carbonates with Chloride under Hydrothermal Conditions

Mercury sorbents M–Al–CO₃ (M=Mg²⁺, Mn²⁺, Fe²⁺, Cu²⁺, or Zn²⁺) were prepared by the coprecipitation of M^{2 +} and Al³⁺ in an alkaline NaOH/Na₂CO₃ solution. The formation of a layered double hydroxide structure significantly enhanced Hg removal, uniquely with a suitable binder as a support. Both Hg(NO₃ )₂ and HgCl₂ were used to perform the capture test. The calcined sorbent exhibited superior efficiency, particularly for materials having bivalent states of mixed oxide (MO), such as calcined Mn and Fe. Chloride ions effectively raised the mercury scrubber efficiency, regardless of the type of sorbent. The results show that synthetic sorbents can provide 96% removal of Hg at 200°C. In addition, a suitable binder such as TiO₂ can be used as a support for dispersing metal oxides, which significantly decreases the MO content while maintaining a high equivalent capture capacity.